Evidence for Internal Desynchrony Caused by Circadian Clock Resetting.

Circadian disruption has been linked to markers for poor health outcomes in humans and animal models. What is it about circadian disruption that is problematic? One hypothesis is that phase resetting of the circadian system, which occurs in response to changes in environmental timing cues, leads to internal desynchrony within the organism. Internal desynchrony is understood as acute changes in phase relationships between biological rhythms from different cell groups, tissues, or organs within the body. Do we have strong evidence for internal desynchrony associated with or caused by circadian clock resetting? Here we review the literature, highlighting several key studies from measures of gene expression in laboratory rodents. We conclude that current evidence offers strong support for the premise that some protocols for light-induced resetting are associated with internal desynchrony. It is important to continue research to test whether internal desynchrony is necessary and/or sufficient for negative health impact of circadian disruption.

Aberrant light directly impairs mood and learning through melanopsin-expressing neurons.

The daily solar cycle allows organisms to synchronize their circadian rhythms and sleep-wake cycles to the correct temporal niche. Changes in day-length, shift-work, and transmeridian travel lead to mood alterations and cognitive function deficits. Sleep deprivation and circadian disruption underlie mood and cognitive disorders associated with irregular light schedules. Whether irregular light schedules directly affect mood and cognitive functions in the context of normal sleep and circadian rhythms remains unclear. Here we show, using an aberrant light cycle that neither changes the amount and architecture of sleep nor causes changes in the circadian timing system, that light directly regulates mood-related behaviours and cognitive functions in mice. Animals exposed to the aberrant light cycle maintain daily corticosterone rhythms, but the overall levels of corticosterone are increased. Despite normal circadian and sleep structures, these animals show increased depression-like behaviours and impaired hippocampal long-term potentiation and learning. Administration of the antidepressant drugs fluoxetine or desipramine restores learning in mice exposed to the aberrant light cycle, suggesting that the mood deficit precedes the learning impairments. To determine the retinal circuits underlying this impairment of mood and learning, we examined the behavioural consequences of this light cycle in animals that lack intrinsically photosensitive retinal ganglion cells. In these animals, the aberrant light cycle does not impair mood and learning, despite the presence of the conventional retinal ganglion cells and the ability of these animals to detect light for image formation. These findings demonstrate the ability of light to influence cognitive and mood functions directly through intrinsically photosensitive retinal ganglion cells.

Differential effects of high-fat diet on glucose tolerance, food intake, and glucocorticoid regulation in male C57BL/6J and BALB/cJ mice.

The C57BL/6J strain of laboratory mice is a popular subject for studies of diet-induced obesity and diabetes given its propensity for developing obesity and glucose intolerance when placed on high-fat diet. High-fat diet leads to much lower weight gain in young adult BALB/cJ mice, which appear to be protected from many of the metabolic effects of high-fat diet observed in C57BL/6J mice. In this report, the effects of diet and timing of feeding on body weight, food intake, glucose tolerance, and stress-induced corticosterone and blood glucose responses were assessed in male C57BL/6J and BALB/cJ mice. Lower glucose tolerance was observed in low-fat diet-fed C57BL/6J than BALB/cJ mice at four times sampled across the circadian cycle. Ad libitum high-fat diet increased the amount of daytime eating behavior and led to impaired glucose regulation in C57BL/6J but not BALB/cJ mice. Restricting food availability to either daytime or nighttime did not prevent overall body weight gain, but restricting feeding to nighttime (but not daytime) did prevent the significant increase in perigonadal fat pad mass produced by high-fat diet in C57BL/6J mice. Baseline corticosterone levels at their typical daily peak near onset of daily activity were blunted in both strains of mice after 8 weeks on high-fat diet, without corresponding differences in baseline glucose levels. Restraint stress-induced increases in corticosterone were exaggerated in C57BL/6J mice on high-fat diet, with concomitant increases in blood glucose. Paradoxically, stress-induced corticosterone responses were even more exaggerated in BALB/cJ mice yet with significantly blunted glucose responses compared to C57BL/6J mice, regardless of diet, indicating that corticosterone does not have equivalent glucogenic effects in young adult male BALB/cJ and C57BL/6J mice on high-fat diet. These results document considerable strain differences that may provide means for elucidating the mechanisms involved in diet-induced obesity, while highlighting the need to consider these strain differences when extending the results of mouse studies toward the human condition.

Effects of high fat diet and chronic circadian challenge on glucocorticoid regulation in C57BL/6J mice.

Both high-fat diet and chronic circadian disruption have been associated with increased incidence of obesity and type 2 diabetes in humans. Chronically elevated glucocorticoids, which have considerable impacts on physiological processes such as intermediary metabolism, inflammation, and fat metabolism, have also been implicated in insulin resistance associated with obesity and diabetes. In this study, the effects of high-fat diet (HFD) or chronic circadian challenge in C57BL/6J mice on basal and stress-induced corticosterone (CORT) and blood glucose levels were assessed. Baseline and stress-induced levels of CORT, insulin and glucose were measured before and after acute restraint stress at 4 different time points across the light-dark cycle (LD) in male C57BL/6J mice maintained for 8 weeks on HFD or regular chow. After 8 weeks on diet, baseline CORT levels in HFD mice were of similar magnitude but more variable than in mice on low-fat diet, rendering their daily fluctuations arrhythmic according to statistical analysis. Baseline glucose measures were unchanged despite significant 3-fold increases in baseline insulin levels in HFD mice at all time points sampled. Restraint stress yielded considerable decreases in insulin levels and increases in CORT and glucose levels that were significantly exaggerated in the early active period in mice on HFD. These results indicate a circadian influence on stress responses after prolonged consumption of high fat diet. In a separate experiment, C57BL/6J mice were subjected to 6 weeks of an alternating light-dark (LD) cycle comprised of 6 h advances and delays of phase every 5 days to keep the circadian system from establishing consistent circadian entrainment, with a control group of mice under a regular 12:12 LD cycle. While body weights were not significantly affected by chronic circadian challenge, the basal CORT rhythm in alternating-LD mice was significantly dampened. Stress-induced CORT in alternating LD were no different from regular LD group with the exception of ZT 18, at which time the stress response was moderately suppressed compared to controls. These results support that high-fat diet may be contributing to health disorders such as obesity and diabetes in a manner different from any effects of chronic circadian disruption.

Duration and timing of daily light exposure influence the rapid shifting of BALB/cJ mouse circadian locomotor rhythms.

Photic entrainment of the murine circadian system can typically be explained with a discrete model in which light exposures near dusk and dawn can either advance or delay free-running rhythms to match the external light cycle period. In most mouse strains, the magnitude of those phase shifts is limited to several hours per day; however, the BALB/cJ mouse can re-entrain to large (6-8hour) phase advances of the light/dark cycle. In this study, we demonstrate that the circadian responses of BALB/cJ mice are dependent on duration as well as timing of light exposure, with significantly larger phase shifts resulting from >6-hour light exposures, yet loss of entrainment to photoperiods of <2-3hours per day or to skeleton photoperiods. Intermittent light exposures of the same total duration but distributed differentially over the same period of time as that of a 6-hour phase advance of the light cycle yielded phase shifts of different magnitudes depending on the pattern of exposure. Both negative and positive masking responses to light and darkness, respectively, were exaggerated in BALB/cJ mice under a T7 light cycle, but were not responsible for their rapid re-entrainment to chronic phase shifting of the light dark cycle. These results collectively suggest that the innately jetlag-resistant BALB/cJ mouse circadian system provides an alternative murine model in which to elucidate the limitations of photic entrainment observed in other commonly used strains of mice.

Potent inhibition of cell proliferation in the hippocampal dentate gyrus of mice by the chemotherapeutic drug thioTEPA.

Antimitotic drugs used in the chemotherapeutic treatment of cancers induce undesirable but unavoidable side-effects from interruption of normal mitotic processes throughout the body. We have examined whether several such drugs capable of penetrating the blood-brain barrier - thioTEPA and 5-fluorouracil - influence the normal process of cell proliferation underlying neurogenesis in the dentate gyrus of the hippocampus of mice. thioTEPA was found to yield a pronounced dose-related inhibition in cell proliferation, while 5-fluorouracil did not. The magnitude of the inhibition paired with a lack of observable impairment of health in mice indicates a suitable experimental model for elucidating the contributions of hippocampal cell proliferation to cognition and behavior.

Circadian variation in mouse hippocampal cell proliferation.

Hippocampal cell proliferation and concomitant motor activity were examined in adult male mice (C57BL/6J) across a 12:12h light-dark cycle. 5-Bromo-2'-deoxyuridine (BrdU) (200 mg/kg, i.p.) was administered at six equally spaced time points across 24h. A significant change in cell proliferation was found in the hilus (light phase>dark phase), but not in the granule cell layer (GCL)/subgranular zone (SGZ). Since it is generally believed that proliferating cells in the hilus and GCL/SGZ give rise primarily to glia and neurons, respectively, these data suggest a possible circadian influence on gliogenesis, rather than neurogenesis.

Circadian clock-controlled regulation of cGMP-protein kinase G in the nocturnal domain.

The suprachiasmatic nucleus (SCN) circadian clock exhibits a recurrent series of dynamic cellular states, characterized by the ability of exogenous signals to activate defined kinases that alter clock time. To explore potential relationships between kinase activation by exogenous signals and endogenous control mechanisms, we examined clock-controlled protein kinase G (PKG) regulation in the mammalian SCN. Signaling via the cGMP-PKG pathway is required for light- or glutamate (GLU)-induced phase advance in late night. Spontaneous cGMP-PKG activation occurred at the end of subjective night in free-running SCN in vitro. Phasing of the SCN rhythm in vitro was delayed by approximately 3 hr after treatment with guanylyl cyclase (GC) inhibitors, PKG inhibition, or antisense oligodeoxynucleotide (alphaODN) specific for PKG, but not PKA inhibitor or mismatched ODN. This sensitivity to GC-PKG inhibition was limited to the same 2 hr time window demarcated by clock-controlled activation of cGMP-PKG. Inhibition of the cGMP-PKG pathway at this time caused delays in the phasing of four endogenous rhythms: wheel-running activity, neuronal activity, cGMP, and Per1. Timing of the cGMP-PKG-necessary window in both rat and mouse depended on clock phase, established by the antecedent light/dark cycle rather than solar time. Because behavioral, neurophysiological, biochemical, and molecular rhythms showed the same temporal sensitivities and qualitative responses, we predict that clock-regulated GC-cGMP-PKG activation may provide a necessary cue as to clock state at the end of the nocturnal domain. Because sensitivity to phase advance by light-GLU-activated GC-cGMP-PKG occurs in juxtaposition, these signals may induce a premature shift to this PKG-necessary clock state.

Chemotherapy drug thioTEPA exacerbates stress-induced anhedonia and corticosteroid responses but not impairment of hippocampal cell proliferation in adult mice.

Cancer patients often suffer long-lasting affective and cognitive impairments as a result of chemotherapy treatment. Previous work in our lab has shown deficits in learning and memory and hippocampal cell proliferation in mice lasting up to 20 weeks following acute administration of thioTEPA. In this study, the effects of thioTEPA in conjunction with effects of chronic stress on depression-related behavior were examined in C57BL/6J mice, 12 weeks following thioTEPA administration. Chemotherapy-treated mice showed a diminished sucralose preference compared to controls that was further exacerbated after 2 weeks of daily restraint stress. This intensifying effect was not observed in the Porsolt forced swim test. Moreover, stress-induced corticosteroid responses were exaggerated in thioTEPA-treated mice. Cell proliferation in the dentate gyrus of the hippocampus was also impaired similarly by prior thioTEPA treatment and by daily restraint stress, with no additive effect. Results suggest that some depression-related impairments may be exacerbated by chemotherapy treatment through altered corticosteroid regulation.

The chemotherapy agent, thioTEPA, yields long-term impairment of hippocampal cell proliferation and memory deficits but not depression-related behaviors in mice.

ThioTEPA is a chemotherapeutic agent used in the treatment of cancers, and more recently has been proposed as a component of high-dose therapy for young patients with recurrent malignant brain tumors. We previously demonstrated a significant dose-dependent reduction of cell proliferation in the dentate gyrus of the hippocampus in mice immediately following a 3-day regiment of thioTEPA. The aim of this study was to evaluate the long-term effects of thioTEPA treatment on hippocampal cell proliferation and potential effects on memory deficit or depression-related behavior in C57BL/6J mice. A 3-day regimen of thioTEPA (10mg/kg/d, i.p.) yielded a significant reduction in cell proliferation immediately after treatment as assessed by BrdU incorporation, and none of the labeled progeny that initially survived the treatment were detectable one week later. Following a 3-week rebound in proliferation following treatment, a significant deficit in proliferation reappeared and persisted for at least 21 weeks following treatment. ThioTEPA-treated mice subjected to an object recognition test 1, 2, 3, 4, 8, 12, 20 or 30 weeks following treatment demonstrated significant memory deficits at 12 and 20 weeks. Mice demonstrated a similar deficit in an object placement test when tested 20 weeks following thioTEPA treatment. However, no observable effects on performance in the Porsolt forced swim test or the tail suspension test were observed in thioTEPA-treated mice. Together, these studies suggest that cumulative long-term negative effects of thioTEPA treatment on proliferation of new cells in the dentate gyrus may contribute to cognitive impairments associated with its use in the treatment of cancer.

Accelerated re-entrainment to advanced light cycles in BALB/cJ mice.

Circadian rhythms in mammals are coordinated by the suprachiasmatic nuclei (SCN) of the hypothalamus, which are most potently synchronized to environmental light-dark cycles. Large advances in the light-dark cycle typically yield gradual advances in activity rhythms on the order of 1-2h per day until re-entrainment is complete due to limitations on the circadian system which are not yet understood. In humans, this delay until re-entrainment is accomplished is experienced as jetlag, with accompanying symptoms of malaise, decreased cognitive performance, sleep problems and gastrointestinal distress. In these experiments, locomotor rhythms of BALB/cJ mice monitored by running wheels were shown to re-entrain to large 6- or 8-hour shifts of the light-dark cycle within 1-2 days, as opposed to the 5-7 days required for C57BL/6J mice. A single-day 6-hour advance of the LD cycle followed by release to constant darkness yielded similar phase shifts, demonstrating that exaggerated re-entrainment is not explained by masking of activity by the light-dark cycle. Responses in BALB/cJ mice were similar when monitored instead by motion detectors, indicating that wheel-running exercise does not influence the magnitude of responses. Neither brief (15 min) light exposure late during subjective nighttime nor 6-hour delays of the light-dark cycle produced exaggerated locomotor phase shifts, indicating that BALB/cJ mice do not merely experience enhanced sensitivity to light. Fos protein was expressed in cells of the SCN following acute light exposure at ZT10 of their previous light-dark cycle, a normally non-responsive time in the circadian cycle, but only in BALB/cJ (and not C57BL/6J) mice that had been subjected two days earlier to a single-day 6-hour advance of the light-dark cycle, indicating that their SCN had been advanced by that treatment. BALB/cJ mice may thus serve as a useful comparative model for studying molecular and physiological processes that limit responsiveness of circadian clocks to photic input.